A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).
(1) Field of the Invention
This invention relates generally to recombinant polypeptides, and polynucleotides encoding those peptides, that arrest proliferating cells in mitosis, and to methods of treating patients with hyperplasias by administering pharmaceutical compositions comprising recombinant polypeptides or polynucleotides.
(2) Description of the Related Art
Cell proliferation in normal cells is regulated by growth factors that signal the cell to re-enter the cell cycle from a state of quiescence. For example, when wounding occurs, growth factors, e.g., platelet-derived growth factor, stimulate fibroblasts to proliferate. Under normal circumstances, withdrawal of growth factors causes proliferating cells to exit the cell cycle and become quiescent. Cancer cells (neoplasia) have lost the ability to exit the cell cycle and proliferate in the absence of growth factors. That is, the cancer cells think that they are receiving growth factor signals. On the other hand, most benign hyperplasias result from the hyperproliferation of cells due to the inappropriate or uncontrolled secretion of growth factors.
Transition across the G2-M border is crucial to advancement through the cell cycle. The G2-M transition is controlled by the activation of the cyclin-dependent kinase cdc2 by the cyclin B. For a review on the regulation of the cell cycle by the cyclin B/cdc2 complex, see Bastians et al., 1999). Basically, levels of cyclin B accumulate during interphase until they reach an optimal level near the end of G2, at which time, cyclin B binds to and activates the kinase cdc2. The active cyclin B/cdc2 complex phosphorylates various molecules that facilitate the entry into mitosis. The level of cyclin B/cdc2 within the nuclei of proliferating cells reach a maximum at metaphase, upon which time cdc2 activates the destruction of cyclin B. Upon the destruction of cyclin B, the cells are then able to undergo anaphase and hence complete the cell cycle.
Diseases of uncontrolled cell proliferation, or hyperplasias, are common health problems today. Examples of diseases of cell over-proliferation include psoriasis, seborrhea, eczema, benign prostate hyperplasia, congenital adrenal hyperplasia, endometrial hyperplasia, squamous cell (vulvular) hyperplasia, sebaceous hyperplasia, Crohn""s Disease, leukemia, carcinoma, sarcoma, glioma, and lymphoma. Current treatment protocols for these diseases are generally toxic and/or ineffective as permanent cures.
Current cancer therapies involve the use of mitotic poisons, such as taxol or colcemid, or radiation to induce cell death in rapidly dividing cells. Such an approach attempts to kill cancer cells at a faster rate than healthy, naturally proliferating cells. Current forms of chemotherapy have severe side affects due to the destruction of healthy tissue, and are therefore a compromise at best.
Current treatments for other hyperplasias, such as benign enlarged prostate or psoriasis and eczema, are largely based upon the use of steroids or retinoids. For example, psoriasis is commonly treated by oral administration of drugs that engage receptors for glucocorticoids, retinoids and vitamin D, i.e., lipid soluble hormones which bind to nuclear receptors. Much of the current research in psoriasis treatment is now focused on the thiazolidinedione class of drugs that bind to the newly discovered peroxisome proliferator-activated receptor gamma. These drugs are administered orally and target multiple cellular processes, therefore leading to potential side effects. For a review on oral steroid-like treatments for psoriasis, see Pershadsingh, H A, 1999 and Ellis, et al., 2000.
Routine topical treatments for psoriasis and/or eczema include steroid creams that are applied to the skin. However, the use of steroids is associated with several side effects including skin thinning, stretch marks and discoloration. Recently, a new cream called PROTOPIC(copyright), which comprises an immunomodulatory drug called tacrolimus (Asakura, et al., 1999, U.S. Pat. No. 5,955,469), has been approved as a topical therapy for eczema. Although the use of a topical immunomodulator may have advantages over the use of steroidal based creams, immunomodulators exert their effects on the immune system. For individuals who are immunocompromised, the use of steroidal creams or creams containing immunosuppressing immunomodulators could possibly have deleterious effects.
Phototherapy, in the form of UVA or UVB irradiation, either used alone, or in combination with other forms of therapy, is a common treatment for inflammatory skin diseases. An important drawback to using ultraviolet light is the heightened risk of skin cancer in the recipient of such treatment. For a review of recent advances in phototherapy, see Simon, et al., 2000.
Many dermatological diseases such as, for example, psoriasis, eczema and seborrhoea, have two major components, inflammation and hyperproliferation. The current standard treatments for these diseases primarily target the inflammation aspect of the diseases.
There have been some efforts in the development of treatments targeting hyperproliferation of cells. Sato et al, have recently reported that the overexpression of platelet-activating factor receptor (PAFR) in transgenic mice leads to epidermal hyperproliferation that resembles psoriasis. Topical application of a cream comprising the PAFR antagonist WEB2086 to the transgenic mice resulted in the suppression of the number of proliferating cells (Sato, et al., 1999)
Benign prostate hyperplasia (BPH), or enlarged prostate, is another economically important disease of cell hyperproliferation. Over 30% of men in their 70s suffer from BPH and, while the growth in and of itself is harmless, it can lead to other serious urogenital disorders. For example, BPH causes problems in urination and can lead to serious kidney disorders, incontinence and impotence. Over 400,000 prostatectomies are performed each year in the United States. Even though surgical prostatectomy can cause bleeding, infection, impotence, retrograde ejaculation and incontinence, it still remains the predominant therapy for BPH.
In addition to the traditional surgical resection procedures, which are used to open the constricted urethral lumen or to remove the entire prostate, several new surgical methods are being developed for BPH. These novel surgical treatments include microwave thermotherapy, transurethral electrovaporization, laser ablation (Bolmsjo et al., 2000, Ohtani et al., 1999, Gilling et al., 1996). Although these treatments are effective toward correcting bladder retention symptoms, they are surgical and as such may increase the chances of secondary complications such as incontinence and impotence.
Other medical treatments for BPH include the administration of finastride (a 5-alpha reductase inhibitor) or alpha-adrenoceptor antagonists (alpha blockers). Finastride is a 4-aza steroid compound that inhibits the conversion of testosterone to dihydrotestosterone (Stoner, 1990). In clinical trials, finastride has been shown to increase urine flow rate by 30% and to decrease prostate size by 18%. However, users of finastride complain of impotence and decreasing libido, suggesting that this drug affects other physiological pathways (Carraro et al., 1996).
Other agents used to treat BPH are alpha blockers. Alpha-blockers are alpa-adrenergic receptor antagonists, which act by relaxing the smooth muscle cells of the prostate, thereby facilitating the flow of urine through the urethra. Additionally, other classes of alpha blockers have been shown to actually suppress prostate growth by inducing apoptosis of prostate epithelial cells. These novel pro-apoptotic effects of some alpha blockers are independent of the alpha-adrenoceptor antagonism (Kyprianou et al., 2000). Side effects of alpha blocker treatment include dizziness, headache, drowsiness and retrograde ejaculation.
The development of safe and effective drugs with specific cell proliferation inhibiting properties would bring significant improvements in the treatment of hyperplasia and cancer. Such drugs can be used alone or in conjunction with current conventional treatments for cancer or hyperplasias, to improve the safety and efficacy of those treatments. This invention is directed to polypeptide and polynucleotide based cell cycle effectors that specifically and narrowly block only the G2/M transition of actively proliferating cells, and the use thereof. By specifically affecting only those cells that have committed to undergo mitosis, the invention would be expected to have little to none side effects. Thus,the development and commercialization of new treatments that safely and effectively target the cellular hyperproliferation aspect of cancer and/or hyperplasia diseases is sorely needed.
(3) References and Related Art
(i) Patent Documents
Asakura et al., Sep. 21, 1999, U.S. Pat. No. 5,955,469. xe2x80x9cPharmaceutical composition.xe2x80x9d
Beach et al., Dec. 9, 1997, U.S. Pat. No. 5,695,950. xe2x80x9cMethod of screening for antimitotic compounds using the cdc25 tyrosine phosphatase.xe2x80x9d
Beach et al., Oct. 5, 1999, U.S. Pat. No. 5,962,316. xe2x80x9cCell-cycle regulatory proteins, and uses related thereto.xe2x80x9d
Beach et al., Oct. 19, 1999, U.S. Pat. No. 5,968,821. xe2x80x9cCell-cycle regulatory proteins, and uses related thereto.xe2x80x9d
Beach et al., Mar. 28, 2000, U.S. Pat. No. 6,043,030. xe2x80x9cCell-cycle regulatory proteins, and uses related thereto.xe2x80x9d
Giordano, Dec. 19, 2000, U.S. Pat. No. 6,162,612. xe2x80x9cHuman cyclin-dependent kinase-like proteins and methods of using the same.xe2x80x9d
Henderson et al., Dec. 16, 1997, U.S. Pat. No. 5,698,443. xe2x80x9cTissue specific viral vectors.xe2x80x9d
Kauffman et al., May 24, 1994, U.S. Pat. No. 5,314,688. xe2x80x9cLocal delivery of dipyridamole for the treatment of proliferative diseases
Wu et al., Jun. 3, 1997, U.S. Pat. No. 5,635,383. xe2x80x9cMethod for the introduction of genes into mammalian cells by a soluble molecular complex comprising a receptor ligand and a polycation.xe2x80x9d
Weiner et al., Jan. 9, 2001, U.S. Pat. No. 6,172,201. xe2x80x9cCellular receptor for HIV-1 Vpr essential for G2/M phase.xe2x80x9d
(ii) Other References
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Carraro et al., 1996, xe2x80x9cComparison of phytotherapy (Permixon) with finestride in the treatment of benign prostate hyperplasia: a randomized international study of 1,098 patients.xe2x80x9d Prostate 29:231-242.
Bastians et al., 1999, xe2x80x9cCell cycle-regulated proteolysis of mitotic target proteins.xe2x80x9d Mol. Biol. Cell 10:3927-3941.
Djavan, B. and M. Marberger, 2000, xe2x80x9cTransurethral microwave thermotherapy: an alternative to medical management in patients with benign prostatic hyperplasia.xe2x80x9d J. Endourol. 14:661-669.
Ellis et al., 2000, xe2x80x9cTroglitazone improves psoriasis and normalizes models of proliferative skin disease: ligands for peroxisome proliferator-activated receptor-gamma inhibit keratinocyte proliferation.xe2x80x9d Arch Dermatol 136:609-616.
Foldvari, et al., 1999, xe2x80x9cDermal and transdermal delivery of protein pharmaceuticals: lipid-based delivery systems for interferon xcex1.xe2x80x9d Biotechnol. Appl. Biochem. 30:129-137.
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Gilbert, S., 1997, Developmental Biology, 5th ed., pp. 200-201, Sinauer, Sunderland, Mass.
Gilling, et al., 1996, xe2x80x9cThe use of the holmium laser in the treatment of benign prostatic hyperplasia.xe2x80x9d J. Endourol. 10:459-461.
King, R., et al., 1996, xe2x80x9cMutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.xe2x80x9d Mol. Biol. Cell 7:1343-1357.
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Ohtani, et al., 1999, xe2x80x9cA new parameter in decision making for transurethral electroresection of benign prostate hyperplasia.xe2x80x9d Eur. Urol. 35:185-191.
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xe2x80x9cPichia expression vectors for constitutive expression and purification of recombinant proteinsxe2x80x9d, Version C, Invitrogen Corporation, Carlsbad, Calif., 2000.
Sato et al., 1999, xe2x80x9cAccelerated proliferation of epidermal keratinocytes by the transgenic expression of the platelet-activating factor receptor.xe2x80x9d Arch Dermatol. 291:614-621.
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Improved treatments targeted toward hyper-proliferative diseases would take advantage of the fact that the disease cells remain in the cell cycle, and therefore target the molecular processes that directly control the cell cycle. The present invention describes a novel, useful and safe composition which is used in the treatment of diseases of cell over-proliferation.
The present invention is based upon the unexpected discovery that a chimeric polypeptide, which is comprised of an N-terminal Tat transit peptide and a C-terminal cyclin B peptide, when added to the liquid media of proliferating HeLa cells, causes the HeLa cells to stop dividing indefinitely (see Example 3). Furthermore, the cells remained healthy in appearance.
One embodiment of the present invention is directed toward polypeptides that arrest eukaryotic cell proliferation. The polypeptide comprises at least a transit peptide domain and an effector domain. The transit peptide domain allows the entire polypeptide to cross the plasma membrane of cells. Transit peptides are well known in the art (Gariepy and Kawamura, 2001).
The effector domain functions to arrest eukaryotic cells in mitosis. In one embodiment of the chimeric polypeptide, the effector domain comprises a portion of a cyclin B or any G2/M cyclin derived from any eukaryotic organism. The cyclin B or any G2/M cyclin may contain amino acid deletions, insertions or substitutions which block the degradation of the polypeptide by the ubiquitin-proteosome complex. In another embodiment of the chimeric polypeptide, the effector domain comprises a portion of a c-Mos polypeptide or any polypeptide that prevents the degradation of a cyclin.
In another embodiment, the invention is directed to an isolated and purified polynucleotide encoding a product which functions to arrest a eukaryotic cell in mitosis. In a prefered embodiment of the polynucleotide, the polynucleotide encodes a portion of a cyclin B, or any G2/M cyclin, under the control of a constitutive or regulatable promoter. The coding region of the cyclin may contain mutations which stabilize or block the degradation of the cyclin mRNA or polypeptide. In another embodiment of the polynucleotide, the polynucleotide encodes a portion of a cMos polypeptide, or any polypeptide which blocks the degradation of a cyclin, under the control of a constitutive or regulatable promoter.
In another embodiment, the invention is directed to a pharmaceutical composition comprising a chimeric polypeptide which arrests eukaryotic cell proliferation. An alternative embodiment of a pharmaceutical composition comprises a polynucleotide encoding a polypeptide which arrests eukaryotic cell proliferation under the control of a constitutive or regulatable promoter.
In another embodiment, the invention is directed to methods of reducing cell proliferation in a patient who suffers from a hyperplasia. The method comprises treating the patient with a pharmaceutical composition comprising a chimeric polypeptide or a recombinant polynucleotide encoding a polypeptide which blocks cell proliferation. In a prefered embodiment, the treatment is directed to patients who suffer from dermal or epidermal hyperplasias such as, for example, psoriasis, eczema, seborrhea, and/or other dermatites. In another embodiment, the treatment is directed to patients that suffer from benign prostate hyperplasia. In another embodiment, the treatment is directed to patients that suffer from cancer, wherein said cancer may be a carcinoma, lymphoma, sarcoma, glioma, myeloma, or leukemia.